The medical industry has become increasingly dependent upon the ability to measure various entities in physiological fluids in order to be able to determine the health status of an individual, dosage level for drugs, use of illegal drugs, genomic sequences, and the like. Thus, the capability of taking a physiological sample and rapidly analyzing for a particular component has made medical therapies more efficient and increasingly successful.
In many instances, one wishes to use plasma as a source to diagnose a patient's health or to monitor the efficacy of drugs that have been administered to the patient. Plasma as a source for the determination of these parameters may have some difficulties. For example, it is well-known in the art that plasma samples containing platelets are still hampered by problems of inaccuracy, lack of precision, and lack of reproducibility. These properties complicate the use of plasma as a sample for diagnostic purposes.
There is, therefore, substantial interest in devising new approaches for using and manipulating plasma for diagnostic purposes. One area of particular interest is the reduction or elimination of platelet interference. When a plasma sample has been properly centrifuged and properly handled after the centrifuging step, a visible upper layer, called the “buffy layer” forms in the sample tube. The buffy layer should contain much of, if not the majority of, the platelets in the sample to minimize platelet interference. Depending upon the centrifugation process, namely length of time of centrifugation and gravitational force, upon completion of centrifugation, the sample will either be a “platelet rich” plasma sample or a “platelet poor” plasma sample. As the terms imply, a “platelet rich” plasma sample contains a significant quantity of platelets in the non-buffy layer portion of the sample as compared to a “platelet poor” plasma sample.
Quantitatively, if the hematocrit (the measure of the volume of red blood cells as a percentage of the total blood volume) of a whole blood sample is approximately 30%, such whole blood sample (i.e., a sample that has not been subjected to centrifugation) can contain as much as about 63% of the number of platelets found in a platelet rich plasma sample. Thus, given a hematocrit of about 30%, platelet rich plasma will comprise about 6.55×105 platelets per microliter of sample, and the whole blood platelet count will be about 4.12×105 platelets per microliter (about 63% of the number of platelets per microliter in the platelet rich plasma sample). The reference range of platelets in whole blood is about 1.5×105 to about 4.5×105 platelets per microliter, which means that a platelet rich plasma sample can have a platelet count of more than 2.0×106 platelets per microliter. A platelet poor plasma sample can have a platelet count of less than 1.0×104 platelets per microliter.
Proper transfer and insertion of the sample tube into the appropriate analytical device after centrifuging minimizes platelet interference in the measurement of the relevant analyte. However, mishandling of sample tubes can occur frequently. For example, the sample may not be centrifuged properly, either being centrifuged for too short a time period or at too low a gravitational force. Alternatively, even if the sample is properly centrifuged, mishandling of the sample tube, e.g., by subsequent agitation or inversion by the human handler, can result in platelets redispersing through the sample, thereby interfering with the measurement of the analyte, and providing a false result. Furthermore, when a false result is suspected, additional tests may be required. This results in wasted resources, both in terms of time and money. Worse yet, a false result may result in severe complications to the patient if the false result is the basis for treatment.
Moreover, for certain STAT assays (i.e., assays that are preferably performed within a short period of time following sample acquisition) such as for hCG and cardiac markers (e.g., CKMB, Troponin I), centrifuging time for the preparation of plasma is sometimes further reduced by clinical laboratories in order to minimize the time that elapses before results can be obtained. With the reduced centrifugation time, the resulting plasma samples will have higher platelet concentrations, which magnifies the platelet interference problem.
Also, as efficiency becomes critical in laboratory environments to reduced cost, increased revenue, and decreased time for providing clinicians with laboratory results, it is greatly desired to have a means by which plasma test samples can be made more “robust”. Plasma samples are more “robust” when they are less susceptible to platelet interference (a) without decreasing efficiency (e.g., without significantly increasing centrifuge time) or (b) without increasing false results (i.e., false positives or false negatives). It can be especially useful and economically valuable if an assay can be conducted more quickly (e.g., by decreasing centrifuging time), while maintaining the robustness of the assay and reducing the incidence of false results. Under ideal circumstances, a false results rate of <5% will be observed with respect to a given assay, but such a low proportion of false results is not normally attained.
Previous attempts have been made to achieve these ends: centrifuging samples, high dilution, addition of anticoagulants, and the like. However, as mentioned above, increasing centrifuge time decreases laboratory efficiency and decreases quality of patient care since it increases the time that is required to obtain test results. Another strategy for addressing this issue involved the addition of anticoagulants to plasma samples, but the results of such strategies have not been favorable. Accordingly, there remains a need for methods that are effective in reducing the incidence of platelet interference in plasma samples, while maintaining or increasing the efficiency of the process of preparing such samples for subsequent analysis.